Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.     

Effects of progesterone antagonist, lilopristone (ZK 98.734), on induction of menstruation, inhibition of nidation, and termination of pregnancy in bonnet monkeys

The effects of a progesterone antagonist, lilopristone (ZK 98.734), on induction of menstruation, inhibition of implantation or pregnancy, and termination of early and mid-pregnancy were studied in bonnet monkeys. In the regularly menstruating animals, administration of lilopristone (25 mg/day, s.c.) during the mid-luteal phase (Days 20-22 of the menstrual cycle) induced menstruation within 2-4 days after the initiation of treatment. A premature drop in circulating progesterone levels was also observed. The luteolytic effect of lilopristone was prevented by exogenous treatment with hCG; however, the animals showed premature menstruation, in spite of high progesterone levels (above 4 ng/ml). Treatment around the time of implantation (between Days 8 and 12 after the mid-cycle peak in estradiol levels) in mated animals provided 100% pregnancy protection. Treatment of pregnant animals on Days 30-32 of the menstrual cycle, i.e. about Day 20 after the estradiol peak, induced abortion in 8 of 10 animals. A significant (p less than 0.05) decrease in serum progesterone levels was observed on Day 3 after the initiation of treatment. However, the decrease was slower (slope: -0.36, r: 0.96) compared to that observed in nonpregnant animals (slope: -0.72, r: 0.95). In the other two animals, pregnancy was not affected. However, when the treatment was delayed until about Day 50 after the estradiol peak, all four animals aborted. This study suggests that lilopristone is a progesterone antagonist with a potential to induce menstruation, inhibit nidation, and terminate pregnancy. The antifertility effects are mediated through blocking progesterone action at the endometrium as well as decreasing progesterone bioavailability, which appears to be due to its effects on gonadotropin release.     

Estrogen and leptin have differential effects on FSH and LH release in female rats

Prior experiments have shown that the adipocyte hormone leptin can advance puberty in mice. We hypothesized that it would also stimulate gonadotrophin secretion in adults. Since the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) is drastically affected by estrogen, we hypothesized that leptin might have different actions dependent on the dose of estrogen. Consequently in these experiments, we tested the effect of injection of leptin into the third cerebral ventricle of ovariectomized animals injected with either the oil diluent, 10 microg or 50 microg of estradiol benzoate 72 hr prior to the experiment. The animals were ovariectomized 3-4 weeks prior to implantation of a cannula into the third ventricle 1 week before the experiments. The day after implantation of an external jugular catheter, blood samples (0. 3 ml) were collected just before and every 10 min for 2 hr after 3V injection of 5 microl of diluent or 10 microg of leptin. Both doses of estradiol benzoate equally decreased plasma LH concentrations and pulse amplitude, but there was a graded decrease in pulse frequency. In contrast, only the 50-microg dose of estradiol benzoate significantly decreased mean plasma FSH concentrations without significantly changing other parameters of FSH release. The number of LH pulses alone and pulses of both hormones together decreased as the dose of estrogen was increased, whereas the number of pulses of FSH alone significantly increased with the higher dose of estradiol benzoate, demonstrating differential control of LH and FSH secretion by estrogen, consistent with alterations in release of luteinizing hormone releasing hormone (LHRH) and the putative FSH-releasing factor (FSHRF), respectively. The effects of intraventricularly injected leptin were drastically altered by increasing doses of estradiol benzoate. There was no significant effect of intraventricular injection of leptin (10 microg) on the various parameters of either FSH or LH secretion in ovariectomized, oil-injected rats, whereas in those injected with 10 microg of estradiol benzoate there was an increase in the first hr in mean plasma concentration, area under the curve, pulse amplitude, and maximum increase of LH above the starting value (Deltamax) on comparison with the results in the diluent-injected animals in which there was no alteration of these parameters during the 2 hr following injection. The pattern of FSH release was opposite to that of LH and had a different time-course. In the diluent-injected animals, probably because of the stress of injection and frequent blood sampling, there was an initial significant decline in plasma FSH at 20 min after injection, followed by a progressive increase with a significant elevation above the control values at 110 and 120 min. In the leptin-injected animals, mean plasma FSH was nearly constant during the entire experiment, coupled with a significant decrease below values in diluent-injected rats, beginning at 30 min after injection and progressing to a maximal difference at 120 min. Area under the curve, pulse amplitude, and Deltamax of FSH was also decreased in the second hour compared to values in diluent-injected rats. In contrast to the stimulatory effects of intraventricular injection of leptin on pulsatile LH release manifest during the first hour after injection, there was a diametrically opposite, delayed significant decrease in pulsatile FSH release. This differential effect of leptin on FSH and LH release was consistent with differential effects of leptin on LHRH and FSHRF release. Finally, the higher dose of E2 (50 microg) suppressed release of both FSH and LH, but there was little effect of leptin under these conditions, the only effect being a slight (P < 0.04) increase in pulse amplitude of LH in this group of rats. The results indicate that the central effects of leptin on gonadotropin release are strongly dependent on plasma estradiol levels. These effects are consistent w     

The ontogeny of immunoreactive, endogenous FSH and LH in the rat ovary during early folliculogenesis

Rabbit antisera to rat pituitary follicle-stimulating hormone (FSH) and to rat luteinizing hormone (LH) were used, in an immunocytochemical probe, to determine the ontogeny and distribution of immunoreactive, endogenous, intraovarian FSH and LH in immature rats. Ovaries from rats 4, 8, 12, and 21 days of age were studied. Both gonadotrophins were first immunodetectable on Day 8. In reactive primordial follicles, LH was restricted to the cytoplasm and nuclei of the surrounding follicle cells. In those follicles possessing both squamous and cuboidal follicle cells, i.e., transitional between primordial and primary, LH was found in both the cytoplasm and nuclei of both follicle cell types. In primary follicles, LH was no longer present in granulosa cells but was concentrated in germ cell cytoplasm. In contrast, in primordial follicles, FSH was restricted to the germ cell but was present in both the oocyte cytoplasm and germinal vesicle. In transitional and primary follicles, FSH remained within the oocyte cytoplasm and germinal vesicle but also became detectable within the cytoplasm and nuclei of granulosa cells. These findings raise some important new questions regarding the role(s) of the gonadotrophins in early follicular development.     

Modulatory actions of the new antiprogestins ZK 98.299 and ZK 98.734 and of RU 486 on luteinizing hormone secretion and progesterone effects in pituitary gonadotrophs

The effects of the antiprogestins (APs) ZK 98.299, ZK 98.734 and RU 486 on GnRH-stimulated LH secretion and their antagonistic activity on progesterone (P) actions were investigated in cultured pituitary cells from adult female Wistar rats. P (100 nM) was able to exert a facilitatory effect on GnRH (1 nM)-induced LH secretion after short-term (4 h) treatment of estradiol-primed (1 nM, 48 h) rat pituitary cells. When the APs (10 pM-10 microM) were introduced during the 4 h incubation period with P the facilitatory effect of P was totally abolished at concentrations greater than 10 nM (ZK 98.299, ZK 98.734) and greater than 1 nM (RU 486). Also the APs were shown to block the inhibitory action of P which occurs after long-term incubation of pituitary cells with this steroid. However at concentrations greater than 10 nM (ZK 98.734, RU 486) and greater than 100 nM (ZK 98.299) this antagonistic action of the APs was lost. To evaluate whether the APs have direct effects on GnRH-induced LH secretion in the absence of exogenous P pituitary cells cultivated for 48 h with or without 1 nM estradiol were incubated for 4 or 24 h with increasing concentrations of the APs (10 pM-10 microM). Four hour treatment of non-estradiol-primed cells with ZK 98.299 or ZK 98.734 was without any effect on the LH response to a 1 nM GnRH-stimulus. Only the highest concentration of RU 486 (10 microM) reduced the LH response. Twenty-four hour treatment of the cultures with the APs led to enhancement of GnRH-stimulated LH secretion by up to 113, 37 and 33% for ZK 98.734, ZK 98.299 and RU 486, respectively. When estradiol-primed cells were used for the same experiments we observed exclusively inhibitory effects on GnRH-induced LH secretion after 4 and 24 h treatment periods. It is concluded that these new APs are potent inhibitors of P-actions, but also per se they induce diverse effects on GnRH-stimulated LH secretion in cultured rat pituitary cells which have to be taken into account.     

Effect of antiprogestin ZK 98.734 on the ovarian cycle, early pregnancy, and on its binding to progesterone receptors in the myometrium of marmoset Callithrix jacchus

The antiprogestin ZK 98.734 (11 beta-(4-dimethylaminophenyl-17 beta-hydroxy-17 alpha-(3-hydroxy-prop-1(Z)-enyl-4,9(10)-estradien-3-one) was administered i.m. (5 mg/day) for three consecutive days to two groups of common marmosets. In one group (nonpregnant, n = 6), it was injected during the luteal phase, and to the second group (pregnant, n = 7), it was injected during early pregnancy, on Days 24-26 of the mid-cycle estradiol peak. Administration of ZK 98.734 during the luteal phase caused a sharp drop in plasma progesterone levels. The luteal phase was shortened whether the drug was administered during the early or the late luteal phase. Similarly, administration of ZK 98.734 during early pregnancy caused a significant drop in progesterone levels, and pregnancy was terminated in all of the animals. The post-treatment cycles in both groups of animals were ovulatory and of normal duration. 3H-ZK 98.734 showed specific binding to myometrial cytosol fraction. ZK 98.734 also displaced the binding of 3H-progesterone to progesterone receptors. However, progesterone had higher binding affinity than did ZK 98.734. The antifertility action of ZK 98.734 could be a result either of its luteolytic action or of its blocking the progesterone receptors in the target tissue. This study, therefore, indicates that in the common marmoset ZK 98.734 is a progesterone antagonist with a potential to terminate early pregnancy.     

Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkeys: a morphological study

Female bonnet monkeys were immunized with 55 kDa porcine zona antigen (ZP-3), with either complete Freund's adjuvant (CFA) or sodium phthalylated lipopolysaccharide (SPLPS) as adjuvant. Anti-ZP-3 antibody titers were monitored and the effect of immunization on the ovarian morphology was assessed by high-resolution light microscopy. The study demonstrated that both adjuvants used were equally potent in eliciting antibody response against ZP-3. Although no morphological damage to ovarian components was observed in animals immunized with SPLPS as adjuvant, immunization using CFA resulted in profound ovarian follicular atrophy, sparing only the primordial follicles. The atrophic phenomenon involved those follicles that either already had or were in the process of forming zona pellucida. The results of this study indicate that choice of adjuvant may be an important consideration for immunization against zona antigens. These findings encourage further investigations for developing better immunization regimen aimed at using zona antigens for immunocontraception.     

Post partum patterns of circulating FSH, LH, prolactin, estradiol, and progesterone in nonsuckling cynomolgus monkeys.

Circulating levels of FSH, LH, prolactin (Prl), estradiol (E), and progesterone (P) were determined by RIA in four intact and four monkeys luteectomized (CLX) at parturition in order to a) characterize the patterns of these hormones during the puerperium, and b) examine a possible inhibitory role of the "rejuvenated" corpus luteum (CL) on the resumption of follicle growth post partum. In both groups during the first four weeks, FSH and LH were at tonic levels typical of ovulatory cycles. Recurrent puerperal "surges" of FSH, but not LH, unaccompanied by increments in serum E, were observed in both intact and CLX monkeys. No consistent pattern of serum Prl was apparent. CLX was followed by a prompt fall in serum P levels, which were elevated above typical follicular phase levels into the second week post partum in intact monkeys. Menstrual cycles resumed 2-4 months after delivery. Hormonal patterns during the first menstrual cycle post partum were indistinguishable from those observed in pregravidic ovulatory cycles. The findings indicate that in nonsuckling cynomolgus monkeys a) although it secretes progesterone, the puerperal CL does not inhibit the resumption of the ovarian cycle post partum, b) the puerperal ovary is not absolutely refractory to gonadotropins, since initial trials with Pergonal + hCG stimulated ovarian function, and c) ovarian activity during the puerperium may be limited by factors other than the tonic supply of gonadotropins.     

Differential effects of LH-RH immunoneutralization on LH and FSH secretion in the ewe

Neutralization of LH-RH by injection of an ovine antiserum to LH-RH in ewes during the late follicular phase of the oestrous cycle resulted in an immediate blockade of pulsatile secretion of LH. Plasma concentrations of FSH gradually rose in the antiserum-treated ewes during the 36-h study period but levels declined in control ewes. These results show that, in the ewe, pulsatile LH secretion is dependent on LH-RH from the hypothalamus, while FSH is largely unresponsive to short-term reduction of LH-RH stimulation. Since reduction in LH secretion is likely to reduce ovarian function, the changes in FSH secretion may be attributed to the removal of a negative feedback influence of an ovarian factor, perhaps oestradiol, on FSH secretion.     

Testosterone selectively increases serum follicle-stimulating hormonal (FSH) but not luteinizing hormone (LH) in gonadotropin-releasing hormone antagonist-treated male rats: evidence for differential regulation of LH and FSH secretion

Both testosterone (T) and gonadotropin-releasing hormone (GnRH)-antagonist (GnRH-A) when given alone lower serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact and castrated rats. However, when graded doses of testosterone enanthate (T.E.) were given to GnRH-A-treated intact male rats, a paradoxical dose-dependent increase in serum FSH occurred; whereas serum LH remained suppressed. This surprising finding led us to ask whether the paradoxical increase in serum FSH in GnRH-A-suppressed animals was a direct stimulatory effect of T on the hypothalamic-pituitary axis or the result of a T effect on a testicular regulator of FSH. To test these hypotheses, we treated adult male castrated rats with GnRH-A and graded doses of T.E. In both intact and castrated rats, serum LH remained undetectable in GnRH-A-treated rats with or without T.E. However, addition of T.E. to GnRH-A led to a dose-dependent increase in serum FSH in castrated animals as well, thus pointing against mediation by a selective testicular regulator of FSH. These data provide evidence that pituitary LH and FSH responses may be differentially regulated under certain conditions. When the action of GnRH is blocked (such as in GnRH-A-treated animals), T directly and selectively increases pituitary FSH secretion.     

LH biosynthesis and secretion in rat anterior pituitary cell cultures: stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [³H]-glucosamine and [³H]-proline into total protein. The incorporation of [³H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [³H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH₂¹⁰]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe², D-Phe⁶]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.     

Plasma concentrations of FSH and LH in entire and castrated prepubertal bull calves treated with Gn-RH.

In bulls there was no increase in plasma FSH and only a small increase in LH over the first 14 weeks of age. In steers (castrated) plasma LH and FSH were unchanged for the first 3 weeks but increased significantly at 7 and 14 weeks. After 100 micrograms Gn-RH, LH release in bulls was minimal until 7 and 14 weeks and there was no comparable rise for FSH. LH and FSH responded to Gn-RH throughout the trial in the steers. The neonatal calf testes selectively inhibited the release of FSH from the pituitary even when challenged with Gn-RH.     

Pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to gonadotropin-releasing hormone during the rat estrous cycle: an increased ratio of FSH to LH is secreted during the secondary FSH surge

The hormonal interactions required for the generation of a secondary surge of FSH on the evening of proestrus have not been clearly defined. The role of GnRH in driving a surge of FSH has been questioned by findings in previous studies. In the current study, gonadotropin secretion was measured from pituitary fragments obtained from rats at 0900 and 2400 h on each day of the estrous cycle. Pituitary fragments were perifused in basal (unstimulated) conditions or in the presence of GnRH pulses to determine whether a selective increase in basal release of FSH and/or an increase in the responsiveness to GnRH occurs during the secondary FSH surge. Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Seven pulses of GnRH (peak amplitude = 50 ng/ml; duration = approximately 2 min) were administered at a rate of one per hour starting at 30 min. Fractions of perfusate were collected every 5 min and frozen until RIA for LH and FSH. The mean total amount of LH or FSH secreted during the hour interval following each of the last six pulses of GnRH (or the corresponding basal hour) was calculated. Analysis of variance with repeated measures indicated that the evening secretion of LH on proestrus (2400 h) dropped significantly (p less than 0.05) from a maximum on the morning of proestrus (0900 h), whereas the FSH secretion remained elevated at this time. Therefore, the ratio of FSH to LH secreted in response to GnRH pulses was highest during the secondary FSH surge and lowest on the morning of proestrus.(ABSTRACT TRUNCATED AT 250 WORDS)